An analysis will be made of the anchorage-independent growth of spontaneously transformed BALB/ c3T3 cells in various stages of transformation. These produce progressively higher efficiencies and larger sizes of colony formation in agar with multiple passaging in cultures. A modified method which maximizes two-dimensional growth of the colonies in agar will be used to study the rates and limitations of multiplication of the various cells. Direct enumeration, recloning and autoradiography will be used to measure multiplication and capacity for further multiplication. Particular attention will be paid to the capacity of cells isolated from agar colonies to produce further colonies in agar, since preliminary results indicate that there is little or no genetic selection for this property, i.e., populations isolated from expanding colonies have the same colony forming efficiency as the initial population. This indicates a great phenotypic heterogeneity in capacity for anchorage-independent growth. Since this property has an extremely high correlation with capacity to produce tumors, the analysis should be relevant to the kinetics of tumor formation. It should also provide a basis for increasing the usefulness of the technique in detecting carcinogenic substances.